Page 406 - IJB-10-1

International Journal of Bioprinting Droplets prepared by air-focused bioprinting

medium (Sigma-Aldrich) supplemented with 10% fetal 2.2.3. Fluidic dynamic simulation of droplets prepared
bovine serum (FBS; Sigma-Aldrich). CAR-T cells targeting by AFMDP
mesothelin (MSLN) were generated by the infection of The formation process of droplets prepared by AFMDP
CAR-encoding lentiviral vector. MSLN-targeting CAR-T was investigated by fluidic dynamic simulation. The
cells were cultured in X-VIVO medium (Lonza, Swiss) microfluidic device model was discretized by the hexahedral
supplemented with 10% FBS, human recombinant IL-15 mesh method. A two-phase axisymmetric numerical
(10 ng/mL, PeproTech, USA), and human recombinant model was developed to simulate the droplet generation
IL-7 (5 ng/mL, PeproTech, USA). The cells were cultured in the two-phase co-flow glass-capillary microfluidic
in a 37 °C incubator (Thermo Fisher Scientific, USA) device. The viscosity of the inner liquid was 2.3 Pa·s, and
under an atmosphere of 5% CO . Cells were sub-cultivated its density was 1.064 g/cm . The viscosity of the outer air
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every 2–4 days at approximately 80% confluence at a split was 0.0000179 Pa·s, and its density was 0.00129 g/cm .
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ratio of 1:3. The inner liquid flow rate was 10 mL/min, and the outer
air flow rate was 200 mL/min. The inner nozzle diameter
2.2. Methods
was 150 μm, and the outer nozzle diameter was 600 μm.
2.2.1. Fabrication of microfluidic devices for AFMDP The parameters used for fluidic dynamic simulations were
To fabricate the glass-capillary microfluidic devices, consistent with those used for experiments.
cylindrical capillaries (inner diameter = 0.55 mm and 2.2.4. Encapsulation and release of CAR-T cells for
outer diameter = 0.96 mm) and square capillaries (inner immune therapy
diameter = 1 mm and outer diameter = 1.4 mm) were To prepare cell-laden hydrogel particles, CAR-T cells
tapered to 150 and 600 μm, respectively, using a pipette were dispersed in 2 wt% alginate solution, which was then
puller. Each tapered cylindrical capillary was inserted emulsified into droplets and collected in a collection bath
into a tapered square capillary, which was then fixed by with 1 wt% CaCl to crosslink the alginate hydrogel. The
epoxy resin. The position of the inner cylindrical capillary 2 wt% concentration of alginate solution was appropriate
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with respect to the outer square capillary could be inward for fabricating hydrogel particles because this viscosity of
contraction, parallel alignment, and outward extension.
Both the inward contraction and the outward extension alginate was easy to crosslink. The air flow rate was 50 mL/
min, and the liquid flow rate was 10 mL/min for CAR-T
distances were 400 μm.
cell encapsulation experiments. Crosslinked cell-laden
Air-focused microfluidic 3D droplet printing system hydrogel particles were washed by DMEM for several
was a modified version of extrusion-based 3D printer. The times and then transferred to the culture medium for cell
microfluidic device was mounted on the printer head of the culture. The cell viability of CAR-T cells encapsulated in
3D printer. The inner capillary of the microfluidic device the hydrogel particles was analyzed on days 1, 3, 7, and
was connected to a syringe pump through a polyethylene 14. Live/dead cells were stained by immersing cell-laden
tube, while the outer capillary was connected to an air hydrogel particles in the medium of a live/dead assay kit
pump with a glass rotameter through a polyethylene tube. for 15 min and imaged at eight different Z-stack slices for
The 3D printing system was used to precisely control the each sample under a confocal microscope. The number
infusion of liquids through the syringe pump and the of live cells stained in green by calcein AM and dead cells
position of printed droplets via programmable codes. stained in red by PI was counted using Image J software.

2.2.2. Preparation of droplets and particles by AFMDP After cell culture, CAR-T cells were released from
Polyethylene glycol was dissolved in deionized water with the hydrogel particles by destroying the 3D crosslinked
a concentration of 5 wt%, 10 wt%, 15 wt%, and 20 wt%. network using PBS solution. To examine the performances
Sodium alginate was dissolved in deionized water with a of CAR-T cells released from the hydrogel particles,
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concentration of 0.5 wt%, 1.0 wt%, 1.5 wt%, and 2.0 wt%. Firefly luciferase-labeled AsPC-1 cells (1 × 10 cells/well)
Liquid was used as the dispersed phase and extruded were cultured with PBS (control group), unencapsulated
through the tapered nozzle of the inner channel, while air CAR-T cells (1 × 10 cells/well), and released CAR-T cells
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was used as the continuous phase and extruded through (1 × 10 cells/well) after culture in hydrogel particles for 2
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the tapered nozzle of the outer channel. A typical liquid weeks, in a 24-well plate. After 48 h, cells were trypsinized,
flow rate was 10 mL/min, while a typical air flow rate was collected, and seeded in a black 96-well plate. To perform
200 mL/min. Dispersed liquid droplets were collected on the bioluminescence-based cell survival assay, D-luciferin
a glass substrate. To prepare alginate hydrogel particles, (RHAWN) was added to the cell suspension with a final
dispersed droplets were collected in a collection bath with concentration of 150 µg/mL and incubated at 37°C for 10
1 wt% CaCl to crosslink the alginate hydrogel. min. The bioluminescent images of live cells were captured
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Volume 10 Issue 1 (2024) 398 https://doi.org/10.36922/ijb.1102